Carboxylic acid reductase (CAR) enzymes belong to the family of oxidoreductases and are specifically categorized under EC 1.2.99.6. They functionally catalyse the reduction of a carboxylic acid (e.g., adipic acid, pimelic acid) to its respective aldehyde, and later alcohols.
Based on sequence homology, each of the known CAR enzymes (EC 1.2.99.6) are composed of two large functional domains, namely an N-terminal Adenylate/AMP/ATP-binding domain and a C-terminal NAD(P)-binding domain, also known as the reductase domain (FIG. 2). The AMP- and the NAD-binding domains enclose a phosphopantetheine attachment site (PP-site) that harbours the phosphopantetheine cofactor which is added post-translationally by a phosphopantetheine transferase (pptase). Catalysis of a carboxylic acid to an aldehyde is postulated to occur by a sequential catalytic mechanism wherein the N-terminal domain catalyzes substrate activation by formation of an initial acyl-AMP intermediate. The intermediate then reacts with the thiol moiety of the phosphopantetheine cofactor to generate a covalently bound thioester. The phosphopantetheine cofactor serves as an arm that swings to the C-terminal reductase domain, which then catalyzes the reduction of the intermediate thioester formed from Acyl-AMP to finish a catalytic cycle. Further analysis of the conserved domain structure by BLAST confirmed the classification of the N-terminal large domain to the Adenylate forming domain (AFD) Class I superfamily, which includes the phosphopantetheine binding site, and the C-terminal large domain belonging to the NAD(P)H/NAD(P)(+) binding superfamily.
In the context of biosynthetic manufacture of nylon intermediates, the requirement for CAR enzyme activity was identified as a rate-limiting step towards the end of the biosynthetic pathway. Therefore, CAR enzymes were selected as candidates for enzyme engineering studies to improve their activity levels.